SERPINA3-ANKRD11-HDAC3 pathway induced aromatase inhibitor resistance in breast cancer can be reversed by HDAC3 inhibition

Endocrine resistance is a major challenge for breast cancer therapy. To identify the genes pivotal for endocrine-resistance progression, we screened five datasets and found 7 commonly dysregulated genes in endocrine-resistant breast cancer cells. Here we show that downregulation of serine protease inhibitor clade A member 3 (SERPINA3) which is a direct target gene of estrogen receptor α contributes to aromatase inhibitor resistance. Ankyrin repeat domain containing 11 (ANKRD11) works as a downstream effector of SERPINA3 in mediating endocrine-resistance. It induces aromatase inhibitor insensitivity by interacting with histone deacetylase 3 (HDAC3) and upregulating its activity. Our study suggests that aromatase inhibitor therapy downregulates SERPINA3 and leads to the ensuing upregulation of ANKRD11, which in turn promotes aromatase inhibitor resistance via binding to and activating HDAC3. HDAC3 inhibition may reverse the aromatase inhibitor resistance in ER-positive breast cancer with decreased SERPINA3 and increased ANKRD11 expression.

Zhou et al. found that SERPINA3 and its target gene ANKRD11 contributed to aromatase inhibitor resistance. The finding is interesting, however, the data in this manuscript cannot fully support the conclusions. Some issues need to be addressed.

1.Why are LTED cells considered as aromatase inhibitors (AI)-resistance cells?
2.Although the level of SERPINA3 was decreased in BC samples after treatment with AI (Fig 2F), and "lower expression of SERPINA3 was specifically related with poor prognosis of ER+ BC (Fig 2G), the data did not support the conclusion that "SERPINA3 is negatively associated with endocrine sensitivity". 5.The efficacy of tamoxifen, aromatase inhibitors or E2 should be observed in AKRD11 overexpressed cells.
6.Knock down AKRD11 in SERPRINAS silencing MCF-7 and T47D cells, and then evaluate the efficacy of E2, tamoxifen, or aromatase inhibitors in these cells.
7. Fig 5f, the effect of "E2 + RGFR966" on proliferation also should be evaluated in control cells and SERPINA3-knockdown cells.
Reviewer #2 (Remarks to the Author): In this work, the authors show that loss of SERPINA3 expression leads to aromatase inhibitor (AI) resistance, and that this resistance is facilitated by ANKRD11 which activates HDAC3 (and hence loss of H3K9Ac). The work is highly relevant and a valuable contribution to the knowledge of AI resistance, and important for future studies of AI resistance in BC. The manuscript is also well written and the logic is easy to follow. Some concerns must be addressed before publication.
Major (more like moderate) concerns In other works (e.g. https://pubmed.ncbi.nlm.nih.gov/25625755/ and others) AI resistance is induced by actual treatment with AIs. Since the target of AIs in postmenopausal women is mainly the adipose tissue and the effect is decreased estrogen concentration in the blood, the strategy applied in the present work makes a lot of sense. However, since this is not established knowledge in the field, the authors should expand their discussion on how estrogen deprivation is representative for AI resistance (and how it may not be). This should be done both upfront (in the introduction) and in the discussion, and comparisons should be made to alternative methods.
The analyses made on patient material are not described in the Materials and Methods. All these analyses must be properly explained. The PDXs are not described, either. The data and analyses used in Figure 2F and 2G should be properly explained and cited in M&M. Even if the data in Figure 2G is used through the bc-GenExMiner, the patient cohort in the analysis should be described and cited. A short summary of patient characteristics in Figure 2F should also be included.
In Figure 2C, data should also be shown for MCF7.
The analysis in Figure 2G for ER positive should also be further stratified into Luminal A and Luminal B, since proliferation could be confounding factor for the survival analysis. This data should be presented in the main text, while the ER negative could be moved to supplementary if needed. Also for figure 4J, the ER negative should be moved to supplementary, and analyses for Luminal A and Luminal B separately should be included.
When investigating how SERPINA3 causes AI resistance, why do the authors focus only on genes negatively correlated to SERPINA3 expression?
Minor: Figure 2D should be transposed (rows and columns), to match fig 2C Figure 3 legend: I) should be H) In the sentence "The data from GSE147271 showed a remarkable downregulation of SERPINA3 and278 up-regulation of ANKRD11 after endocrine treatment ( Fig. 4I)" I would remove "remarkable". It's non-quantitative, and quite subjective.
Reviewer #3 (Remarks to the Author): This paper aimed to demonstrate SERPINA3-ANKRD11-HDAC3 pathway involved in aromatase inhibitor resistance and reversed in HDAC3 inhibition. The experiments were well-organized in two ERpositive cell lines. However, there are concerns about the cell lines used in the study, which are the main models to exhibit the resistant mechanism in the study. Thus, please provide more details on how the author characterized LTED cell lines and how to demonstrate that these cell lines can mimic AI-resistance.

Authors' Reply
We sincerely thank you for giving us the opportunity to revise the manuscript, and we deeply appreciate the insightful and constructive comments from the reviewers. In this updated manuscript, we have thoroughly revised the manuscript according to the reviewers' comments. Please see below our point-by-point responses. The finding is interesting, however, the data in this manuscript cannot fully support the conclusions. Some issues need to be addressed:

Author's reply to the Reviewers
We sincerely thank the reviewer for these valuable comments.

Why are LTED cells considered as aromatase inhibitors (AI)-resistance cells?
In clinical practice, aromatase inhibitors (AIs) are typically administered in postmenopausal breast cancer patients to inhibit estrogen synthesis in the peripheral tissues such as adipose tissue, breasts and skin, thereby decrease blood estrogen concentrations and prevent cancer cell growth (Ma CX, et al. Nat Rev Cancer. 2015), but not directly act on the cancer cells themselves because of low or no expression of endogenous aromatase in cancer cells (Chen X, et al. Nat Commun. 2022), which could be manifested by the insensitivity of ER+ breast cancer cells to therapeutic blood concentrations of the letrozole (50-100 ng/ml), anastrozole (10-40 ng/ml) and exemestane (10-20 ng/ml) (Awada A, et al. Eur J Cancer. 2008;Valle M, et al. Br J Clin Pharmacol. 2005;Gervasini G, et al. Br J Clin Pharmacol. 2017) (Supplementary Fig. S3). Long-term-estrogen-deprivation (LTED) model was established by maintaining ER-positive breast cancer cells in estrogen deprivation condition for over one year to more closely mimic the physiological estrogendepletion situation induced by AI therapy in patients than other models. The validity of LTED model in mimicking AI-resistance has been widely accepted, as evidenced by the use of this model in a number of high-quality AI resistance studies (Chen X, et al. Nat Commun. 2022;Williams MM, et al. Cell Death Dis. 2018;Du T, et al. Breast Cancer Res. 2018;Bhola NE, et al. Cancer Res. 2015;Fox EM, et al. Cancer Res. 2011;Miller TW, et al. J Clin Invest. 2010). In 2. Although the level of SERPINA3 was decreased in BC samples after treatment with AI ( Fig 2F), and "lower expression of SERPINA3 was specifically related with poor prognosis of ER+ BC (Fig 2G), the data did not support the conclusion that "SERPINA3 is negatively associated with endocrine sensitivity".
We thank the reviewer for pointing out this issue. In light of this advice, we changed the original sentence "Furthermore, lower expression of SERPINA3 is specifically related with poor prognosis of ER+ BC, but not ER negative (ER-) subtype according to the bc-GenExMiner v4.8 database (Fig. 2g), suggesting SERPINA3 expression is negatively associated with endocrine sensitivity in ER+ BC." to the revised sentence "Furthermore, according to the data retrieved from TCGA database (Supplementary Table. (Fig. 2g), but not in ER-subtype ( Supplementary Fig. S1). All above results together suggest that the expression of SERPINA3 is negatively associated with endocrine resistance in ER+ BC." As demonstrated in the revised sentence, we want to express that all results in Fig.2, including that SERPINA3 knockdown endowed breast cancer cells with estrogen deprivation resistance ( Fig. 2b and c), while SERPINA3 overexpression restored endocrine sensitivity in LTED cells (Fig. 2d), and the observed diminished expression of SERPINA3 in LTED cells compared with their parent counterparts (Fig. 2e), as well as Fig. 2f and g mentioned in the question, jointly indicate that "SERPINA3 is negatively associated with endocrine resistance".  Fig. 3, support our conclusion that SERPINA3 expression was inhibited by ERα pathway. can also be an intractable problem to transform such a large plasmid into competent cells (Li M, et al. Microb Cell Fact. 2012). Therefore, we are sorry that only loss-of-function studies were used to demonstrate the positive role of ANKRD11 in estrogen-deprivationresistance development.
6. Knock down AKRD11 in SERPRINAS We appreciate this constructive suggestion. As suggested, to observe  and T47D cells (Fig. 4d)." (Page 16, Lines 315-317).   We sincerely apologize for the negligence and thank the reviewer for pointing out. Revisions have been made accordingly. A "Data All these analyses must be properly explained. The PDXs are not described, either. The data and analyses used in Figure   2F and 2G should be properly explained and cited in M&M. Even if the data in Figure 2G is used through the bc-GenExMiner, the patient cohort in the analysis should be described and cited. A short summary of patient characteristics in Figure 2F should also be included.
retrieval" section has been added in the Materials and Methods in the revised manuscript (Pages 7-8, lines 114-135). And "Statistics and reproducibility" section in the Materials and Methods was also introduced in more detail (Pages 11-12, lines 219-227). The references that provide the original expression profiling data were accordingly cited (Reference 40-42). Brief characteristics description on patients in GSE105777, GSE153470 and GSE147271 was also included in figure legends.
3. In Figure 2C, data should also be shown for MCF7.
As recommended, the effect of SERPINA3 knock-down on estrogen deprivation resistance was examined in MCF-7 using colony formation assay, as such in T47D. The result was shown in Fig. 2c.
Manuscript was modified accordingly at Page 13, lines 260-262: Plate colony formation assay performed in MCF-7 and T47D cells confirmed that knock-down of SERPINA3 expression would result in E2-deprivation-resistance (Fig. 2c).  First we would like to express our sincere gratitude for the reviewer's careful and thorough review on this manuscript. We appreciate these corrections very much.
6. Figure  We appreciate the reviewer's concern about the rationality of LTED ( Fig.1a and b) and tamoxifen (TAM) treatment ( Fig.1c and d) on their growth and survival. Please refer to Pages 12-13, lines 237-243 in revised manuscript for detailed description. In addition, the expression of ERα was shown to be downregulated in both LTED cells by QPCR ( Fig.3c) and western blot (Fig. 3d) when compared with their parent cells.  updated manuscript, we have revised the manuscript according to the reviewer's comments and format requirements. Please see below our response to the reviewer #3. We sincerely thank the reviewer for doing an in-depth reading of our work and providing this valuable comment. And we hope the answer below could meet the reviewer's concerns.

Author's reply to the Reviewers
The LTED model, which has been proven to resistant to low plasma E2 concentration in vivo [Shim WS, et al. Endocrinology. 2000], was established by long-term estrogen deprivation. Therefore, the cells developed in this process are results of estrogen deficiencyresistance (or AI resistance) screening, and naturally are also resistant to tamoxifen that works by blocking E2 signaling. Two other reports showed similar findings that tamoxifen resistance was also identified as a property of LTED cells [Martin LA, et al. Endocr Relat Cancer. 2005;Henriques Palma GB & Kaur M. Anticancer Res. 2022].
However, to avoid ambiguity or misinterpretation that may arise in the tamoxifen resistance of AI-resistant LTED cells, we removed the tamoxifen part from Figure 1.